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THE  EFFECT  OF  11  ME  AND  TEMPERA- 
TERE  UPON  THE  BROMINE  VALUE 
OF  FISH  OILS 


MY 


VIVIAN  JONKS  YAl.E 


THESIS 

FOK  THE 


DEGREE  OF  BACHELOR  OF  SCIENCE 


IN 

LIBERAL  ARTS  AND  SCIENCES 


COLLEGE  OF  LIBERAL  ARTS  AND  SCIENCES 


UNIVERSITY  OF  ILLINOIS 


1922 


-.V . 

eiOV1UJt*qO  7Tl*«SV»!<W  ' W' 


•Vi 

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i?;i  . r4:tXX».  «' 


TABLK  OF  COTv^TF^TTB. 


A 3 kn  o wl  e dgrn  en  t p ag  e 

Introduction  1 

Historic  3,1  u 

Theoretical  4 

Experimental  9 

Results — Table  for  Cod  Oil  16 

Table  for  Herring  Oil  16 

Table  for  Salmon  Oil  17 

Ta'^le  for  Sardine  Oil  17 

Table  for  Japanese  Herring  Oil  18 

Table  for  Menhaden  Oil  18 

Conclusions  25 

Bibliography  26 


Digitized  by  the  Internet  Archive 
in  2015 


https://archive.org/details/effectoftimetempOOyale 


ACKNOWLEDGMENT. 


I wish  to  express  my  sincere  appreciation  of 
the  valuable  assistance  vihich  has  been  received  during  this 
investigation,  from  Dr.  George  D,  Beal,  under  whose  direction 
it  was  carried  out. 


INTRODUCTION. 


Natural  oils  and  fats  are  widely  distributed  throughout 
the  vegetable  and  animal  kingdon,  from  the  lowest  organisms  up  td 
the  most  highly  organized  forms  of  vegetable  and  animal  life,  and 
are  found  in  almost  all  tissues  and  organs. 

In  plants  they  are  chiefly  found  in  the  seeds  and  are 
associated  therein  with  the  starch  to  nouri^  the  embryo.  In  the 
animal  organisms,  the  oils  and  fats  are  mainly  enclosed  in  the  cel 
Lular  tissues  of  the  intestines  and  in  the  tissues  nearest  the 
outer  skin. 

Considered  chemically,  fats  and  fatty  oils  consist 
chiefly  of  glycerol  tri-esters  (glycerides)  of  saturated  fatty 
acids  a.nd  of  related,  unsaturated  acids. 

The  properties  of  the  fats  run  parallel  with  those  of 
the  fatty  acids  and  since  this  paper  will  deal  with  the  unsat- 
urated fatty  acids  a few  of  their  properties  will  be  considered 
kiere. 

These  acids  contain  one  or  more  pairs  of  carbon  atoms 
united  by  a double  bond  and  are  therefore,  capable  of  taking  up, 
oy  direct  addition  atoms  of  halogen  or  halogen  acid  to  form 
saturated  compounds;  the  quality  of  the  halogen  absorption  being 
dependent  entirely  upon  these  double  unions. 


HISTORICAL. 

In  about  the  year  1857  Cailletet'  communicated  to  the 
Societe  Industrielle  de  Mulhouse  a titration  process  for  the 
determination  of  the  bromine  absorption  value  of  an  unsa  tur- 

ated  f atty  acid,  to  be  thus  performed: 

"To  any  given  oil  add  a five  per  cent  aqueous 

solution  of  caustic  potash  and  agitate;  then  pour  in  an  excess 
of  a 33  1-3  per  cent  alcoholic  solution  of  bromine;  lastly, 
add  a two  per  cent  solution  of  turpentine  until  the  color  of  the 
free  bromine  is  completely  discharged.  The  turpentine  solution 
is  supposed  to  be  known  in  terms  of  the  bromine  solution,  and 
thus  the  bromine  absorption  of  any  oil  can  be  determined.  If 
stand  ard  pure  oils  be  at  hand,  their  admixture  can  be 
quantitatively  ascertained  by  this  means." 

Cailletets'  process  had  some  very  obvious  disadvantage 
according  to  E,  J,  Mills^,  the  solution  of  bromine  can  not  be 
preserved  unchanged,  probably  even  for  a few  hours;  and  the 
presence  of  water  and  alcohol  must  inevitably  tend  to  promote 
oxidation  of  the  oil  under  treatment.  Again,  Cailletet’  admits 
that  the  absorption  increases  in  total  amount  with  so  moderate 
a rise  of  temperature  as  from  10°  to  ZOP  c,  and  that  a consid- 
erable amount  of  bromination  occurs.  It  occurred,  accordingly, 

to  Mills^  (1379-1880),  more  particularly  in  connection  with 
Hydrocarbon  oils,  that  weak  aqueous  bromine  alone  might  give 


-3- 


gentler  and  steadier  results. 

This  plan  v/as  accordingly  tried  with  various  samples 

of  petroleum,  the  sample  being  agitated  with  100  parts  of  water, 

and  a one-tenth  per  cent  solution  of  bromine  added,  with  constant 
agitation,  until  potassium  iodide  and  starch,  used  as  an 
external  indicator,  gave  a "^lue  color.  Under  these  circumstances 

constant  bromine  absorptions  were  obtained  accompanied  always 

with  some  bromine  absorption.  The  chief  difficulty  in  applying 

this  otherwise  satisfactory  method  consisted  in  the  extreme 

amount  of  technique  required  to  obtain  uniform  measurements. 

(1831)  subsequently  st^'gested  agitating 
0 s with  excess  of  aqueous  sodium  hypobromite  and  hydrochloric 

aoid,  and  titrating  the  ultimate  free  bromine.  This  amounts 

to  treatment  with  nascent  as  well  as  free  bromine  in  the 

presence  of  water;  so  that  the  results  are  of  a very  composite 
character. 

As  it  was  evidently  desirable  to  confine  the  absorption 
to  the  formation  of  additive  and  not  substitution  products,  it 
was  decided  to  employ  CS3  as  the  common  solvent  of  the  bromine 
and  the  substance  to  be  titrated.  Water  was  thus  practically 

excluded  and  the  reaction  was  marked  by  great  regularity  and 
considerable  sharpness. 

Mills  later  substituted  CCI4  explaining  the  change 

by  saying  that  at  ordinary  temperature  the  solution  of  ©romine 
is  more  stable  in  CCl^  than  in  CS3.  Thus  it  is  to  him  that  we 
owe  the  application  of  the  bromine  absorption  value  to  the 
analysis  of  fats.  His  method  is  as  follows: 


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-4- 


.1  gram  of  oil  or  fat  is  tried  tnoroughly  and  filtered, 
then  dissolved  in  50  cc  of  CCla  and  placed  in  a narrow-mouthed 
stoppered  hottle  of  100  cc  . capacity.  To  this  solution  is 
added  a standard  carbon  tetrachloride  solution  (about  .006- 
.008  grams  per  c .c  . ) until  after  a lapse  of  fifteen  minutes, 
a coloration  persists.  The  excess  of  bromine  can  be  measured 
either  by  this  color  compared  to  that  produced  by  a blank 
experiment  or,  more  accurately  by  titrating  back  with  a standard 
solution  of  B-napthol  in  carbon  tetrachloride,  when  monobrom 

naphthol  is  formed.  The  bromine  is  calculated  to  -t,bsorb  100  grams 
of  fat . The  average  probable  error  is  said  to  be  about  .46 
per  cent  . 

Mills  emphatically  stressed  the  fact  that  moisture  must 
be  rigidly  excluded,  since  in  its  presence  the  bromine  absorption 
number  was  found  to  be  too  high.  Instead  of  B-naphthol,  potassium 
iodide  may  be  added,  and  the  liberated  iodine  titrated  with 
standard  sodium  thio-sulf ate  . 

THEORETIC  L . 

The  bromine  (or  iodine)  absorption  value  is  defined 
as  the  per  cent  of  bromine  (or  iodine)  absorbed  by  the  sample . 

This  value  is  a quantitative  measure  of  the  proportion  of 
unsaturated  fatty  acids,  which,  both  in  their  free  state  and  in 
combination  with  glycerol,  have  the  property  of  assimilating 
halogens  to  form  saturated  compounds. 


#5* 


Two  factors  influence  the  result; 

(l)'  the  nature  of  the  unsaturated  acid  yj  hich  is  present, 

those  with  two  doubl  e bonds  absorbing  more  than  those 
with  one  even  though  the  number  of  carbon  atoms  is  the 
same,  and 

(3)  the  chemical  composition  of  the  glycerides,  those  with 
the  lower  molecular  weights  absorbing  a greater 
percentage  than  those  with  higher,  where  the  degree  of 
saturation  is  the  same. 

It  is  hardly  possible  to  allow  dry  bromine  to  act  on  an 
oil  since  it  is  absorbed  with  a more  or  less  violent 
reaction  and  hydrobromic  acid  is  evolved,  hence  the 
reaction  m.ust  be  moderated  by  previously  dissolving 
both  the  bromine  and  the  oil  in  a suitable  solvent . 

Carbon  tetrachloride,  as  was  first  used  by  Mills®  is  generally 
employed  as  the  solvent.  In  the  course  of  the  reaction,  even 
when  carbon  tetrachloride  is  used,  a small  q uantity  of  hydro- 
bromic is  formed,  due  to  the  substitution  of  hydrogen  by  bromine 
in  the  molecule  of  the  fatty  substance.  Therefore  the  total 
absorption  of  bromine  is  due  to  both  substitution  and  addition, 
and  since  greater  amounts  of  substitution  products  are  formed  wjJh 
more  concentrated  brom.ine  solutions,  moderately  dilute  solutions 
should  be  used.  The  amount  of  substitution  which  has  taken  place 
can  be  determined  by  a measure  of  the  hydrobromic  acid  formed  and 
the  true  bromine  a bsorption  value,  will  be  the  total  absorption 


-6- 


value  minus  the  bfomine  reacting  by  substitution. 

Mcllhiney'^  in  1894  determined  the  bromine  addition  and 
the  substitution  values  as  follows: 

^Dissolve  from  .35  to  1 gram  of  the  oil  or  fat  in  10  cc 
of  carbon  tetrachloride  in  a 500cc  stoppered  bottle  and  add  an 
excess  of  one-third  normal  solution  of  bromine.  Place  in  ice 
after  a few  minutes  in  order  to  create  a partial  vacuum.  Next 
slip  a piece  of  rubber  tubing  over  the  neck  to  form  a well  and 
through  this  suck  35  cc  water  into  the  bottle.  The  contents  are 
well  shaken  and  about  1 0 cc  of  30  percent  potassium  iodide  e.nd 
about  75  cc  of  v/ater  added.  The  iodine  liberated  is  measured  by 
titrating  it  with  a standard  solution  of  sodium  thiosulfate  and 
then  calculated  to  bromine.  The  total  amount  of  bromine  added  is 
ascertained  by  a blank  test,  and  the  difference  betw'een  the  two 
equals  the  to  tal  bromine  absorption.  This  is  calculated  to  units 
in  per  cent  of  the  sample  taken.  The  contents  of  the  bottle  are 
now  transferred  to  a sep'aratory  funnel,  and  the  aqueous  solution 
separated  and  filtered.  If  the  filtrate  is  blue,  it  is  decolorize! 
by  sodium  thiosulphate  and  the  free  acid  is  determined  as  hydrobro- 
mic  acid  by  titration  w ith  decinormal  alkali,  methyl  orange  being 
the  indicator.  The  bro  mine  calculated  from  the  hydrobromic  acid 
and  expressed  in  per  cent  of  the  sample  gives  the  bromine 
substitution  value,  Tw  ice  this  number  subtracted  from  the  total 

bromine  absorption  value  furnishes  the  bromine  addition  number. 

In  order  to  have  a standard  by  which  to  measure  the 

experimental  portion  of  this  paper,  the  iodine  values  of  the  oils 


I 


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4 


-7- 


under  exa  mination  were  first  determined.  There  are  three  methocb 
at  present  which  are  most  commonly  used  for  the  determination  of 
the  iodine  absorption  value,  those  of  H-Cibl,  Wijs  and  Hanus. 

The  first  (Httbl)^  consists  in  the  main  in  s.dding  50  cc 
of  the  iodine  solution  (consisting  of  a mixture  of  26  grams  of 
pure  iodine  in  500  cc  of  95  per  cent  alcohol  and  30  grams  of 
mercuric  chloride  in  500  cc  of  alcohol,  mixed  in  equal  volumes 
and  let  stand  12  hours)  t6  the  sample,  which  is  dissolved  in 
chloroform  in  a special  Erlenmeyer  flaslfc.  The  flask  is  stoppered 
tightly  and  the  gutter  filled  with  fifteen  per  cent  potassium 
iodide,  then  allowed  to  stand  three  hours — a blank  being  run  at 
the  same  time — then  20  cc  more  potassium  iodide  is  added  and  the 
contents  titrated  v/ith  thiosulfate.  The  absorbed  iodine  is  cal- 
culs.ted  in  terms  of  percentage  of  the  weight  of  the  sample. 

This  method  has  been  proven  to  be  one  of  the  most 
valuable  ones  to  be  used  in  the  technical  analysis  of  oils  and 
fats.  Long  before  any  detailed  explanation  had  been  found  for 
the  reactions  involved,  the  numbers  obtained  afforded  a valuable 
guidance  in  the  examination  of  oils. 

Experiments  by  Wijg  led  to  a satisfactory  explanation 
of  the  reactions  occurring  in  the  Hftbl  solution.  The  important 
change  which  takes  place  on  mixing  the  solutions  of  mercuric 
chloride  and  iodine  is  thus  represented: 

HgCl3  +■  2 12  «=  Hgip.  4 g ICl 


K 


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During  standardization  potassium  iodide  solution  and 
water  are  added  and  the  following  reaction  occurs, 

ICl  ♦ KI  * Kci  4-  Is 

Thus  it  is  to  he  discerned  that  provided  no  other  reaction  has 
taken  place,  all  of  the  iodine  originally  employed  must  he  found 
in  the  hi  ank  test  as  iodine.  Wijs'  method  differs  from  that  of 
Kfiihls*  only  in  that  his  iodine  solution  is  prepared  hy  dissolving 
13  grams  of  iodine  in  a liter  of  glacial  acetic  acid  and  then 
passing  in  chlorine  gas  until  all  is  converted  into  iodine  chlor- 
ide. This  amounts  practically  to  a tacit  acceptance  that  iodo- 
chloride  is  the  active  agent. 

Hanui^  suggested  that  Wi^s’  method  he  modified  hy 
using  iodine  bromide  instead  of  iodine  chloride.  In  this  method 
the  oil  is  dissolved  in  10  cc  of  chloroform  and  35  cc  of  the 
iodine  solution  is  added.  The  mixture  is  allowed  to  stand  for 
some  time  with  occasiona,!  shaking  and  then  1 0 cc  of  a 15fj 
solution  of  potassium  iodide  and  100  cc  of  water  are  added  and 
the  excess  iodine  titrated  vfith  standard  thiosulphate  until  the 
blue  color  obtained  with  the  aid  of  starch  paste  just  fails  to 
reappear . 

The  results  of  this  method  agree  very  closely  with  those 
obtained  hy  Hiihls’  method  but  the  results  obtained  by  the  Wijs 
method  are  higher  than  the  others. 

EXPERIMENTAL . 


-9- 


EXPERIMENTAL . 

The  experimental  portion  of  this  paper  represents  an 
attempt  to  determine  the  conditions  necessary  for  the  accurate 
determination  of  the  bromine  absorption  values  of  fish  oils.  Tbe 
oils  used  for  the  work  of  this  paper  were  Cod  Oil,  Salmon  Oil, 
Herring  Oil,  Sardine  Oil,  Japanese  Herring  Oil  and  Menhaden  Oil. 

The  solutions  used  in  the  determination  of  the  bromine 
absorption  values  were  prepared  as  follows; 

1.  Standard  potassium  permanganate,  used  for  the 
standardization  of  the  thiosulphated  solution.  The 
potassium  permanganate  was  standardized  against  chemically 
pure  sodium  oxalate  in  the  usual  manner. 

I,  II.  III. 

Wt . of  Sample  .1843  .1467  .3031 

CCS  Mn04  3^6  23.6  33.5 

NF.  KMno4  .0939  .0938  .0933 

Average  Normality  Factor  .0930 
3.  Sodium  P^*^^sulphate,  standardized  against  the 
potassium  permanganate  as  follows: 

Approximately  10  cc  of  15  per  cent  potassium  iodicfe 
was  placed  in  a glass-stoppered  flask,  then  10  cc 
potassium  permanganate  solution  vir  ere  added  from 
a burette,  followed  by  ten  cc  dilute  hydrochloric 
acid;  and  the  solution  allowed  to  stand  ten 
minutes . 


-10- 


One  hundred  cc  E^O  were  then  added  and  the  solution  was  titrated 
with  the  sodium  thiosulphate-starch  being  used  as  the  indicator. 
The  results  were  as  follows  for  the  three  standard 

solutions: 


I . II . 

(a)  No.  CCS  KI«'Ino4  10.  10. 

" " Na2Ss03  9.92  9.9 

N.  F.  Na^SgOs  .0937  ■ .0939 

Average  Normality  Factor  .0938 

I.  II. 

(b)  No.  CCS  KMr.04  10.25  10. 

" " NagSoOs  10.05  9.8 

N.  F.  Na3S30;3  .0948  .0948 

Average  Normality  Factor  .0948 


III, 

10. 

9.92 

.0937 


(c)  I.  II. 

No.  CCS  KMno4  10.  10. 

" " NagSgOs  9.4  9.4 

N.  F.  NagSgOs  .0989  .0989 


Average  Normality  Factor  .0989 
3,  Bromine  solution — prepared  by  diluting  5 1-3  cc 


or  approximately  16  grams  of  bromine  with  enough  carbon 
tetrachloride  to  make  1 liter  of  solution. 

4.  Potassium  Iodide — 150  gras,  to  the  liter. 

5.  Starch  solution  prepared  by  boiling  0.5  grams  of 
soluble  starch  with  100  ccs.  water. 


b -11- 

The  following  additional  solution  was  used  for  the 
determination  of  the  iodine  values: 

6.  Hanus  solution — 13.3  grms . iodine  were  dissolved 
in  1000  cc.  of  glacial  acetic  acid  and  enough  bromine — 
about  3 cc — added  to  double  the  halogen  content. 

In  order  to  establish  a standard  for  the  determined 
bromine  absorption  values — and  hence  iodine  values  calculated 
from  them — the  iodine  values  of  the  six  oils  were  first  determined, 
according  to  the  Hanus  method. 

(see  following  page) 


Menhaden  .3146  25  28.1  75.4  .0938  178.95 

Average  178.79 


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Oil  Sample  reagent  Ccs  NagS202  blank  N.  F.  i\*a2S203  Iodine  No 

Cod  .2704  25  42.08  75.68  .0938  147.92 


( 


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-13- 

The  bromine  absorption  va,lues  of  the  oils  were  obtained 

as  follows: 

From  .3  to  ,3  grams  of  oil  were  weighed  into  a flask 
with  a well  fitting  glass  stopper,  10  cos  carbon  tetrachloride 
were  then  added  in  order  to  dissolve  the  oil  and  then  35  ccs  of 
the  bromine  solution,  and  the  flask  packed  in  ice  and  salt  for 
varying  lengths  of  time  as  shovm  belov/. 

A blank  v/as  run  for  every  group  of  samples,  being  treated 
exactly  the  same  as  the  samples.  At  the  end  of  the  specified  tim.e, 
the  flasks  were  removed  from  the  container,  15  ccs  of  a fifteen 
per  cent  solution  of  potassium  iodide  and  100  ccs  of  water  were 
added  and  the  m.ixture  titrated  at  once  v/ith  sodium  thiosulphate, 
starch  being  used  as  an  indicator. 

With  the  exception  of  the  Menhaden  oil,  very  little 
trouble  was  encountered  in  securing  good  results.  The  am.ateur 
is  apt  to  shake  the  mixture  too  vigorously — and  perhaps  he  may 
wait  just  too  long  before  adding  the  indicator — but  after  a certain 
technique  is  secured  the  titration  becomes  quite  accurate  and  the 
endpoint  is  easily  discerned. 

After  the  liberated  iodine  had  been  titrsvted  with  tenth 
normal  sodium  hydroxide,  using  phenol thalein  as  the  indicator. 
Practically  no  variation  occurred  in  the  amount  of  alkali  used  so 
it  was  shown  that  very  little  substitution  was  taking  place  and 
hence  it  was  deemed  unnecessary  for  the  most  accurate  to  calculate 
this . 


\ i 


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-14- 

In  order  to  study  the  length  of  time  necessary  for  the 
most  accurate  determination  of  the  bromine  absorption  value,  as 
compared  with  the  iodine  value,  determinations  were  made  in  the 
following  series  one  hour,  two  hours,  four  hours,  eight  hours, 
and  sixteen  hours.  The  temperature  was  that  of  an  ice  and  salt 
mixture  and  varied  from  -10^  C to  -16*^  C-being  kept  constant  in 
each  case  by  mieans  of  a heavily  insulated  box  in  which  the  samples 
were  surrounded  by  the  freezing  mixture. 

The  results  obtained  were  calculated  to  the  percentage 
of  bromine  absorbed,  or  the  number  of  grams  of  bromine  v/hich 
lOG  grams  of  fat  v\Tould  absorb;  then  from  this  the  Iodine  number 
was  calculated  and  compared  with  the  theoretical  value  for  the 
Iodine  number. 

The  following  explains  the  determination  and  calculations 


involved . 

Cod  Oil. 

First  V/t . 

Second  Wt . 

Sample  Wt . 

Ccs  Bromine  solution 
Ccs  llagSsOs 
Ccs  Na2Sg02  blank 
N.  F.  NagSgO^ 

T ime 
Br . No. 


47.4377 
47  .2077 
.2300 
25. 

35. 

61.3 
.0948 
8 hours 
86.52 


Calc.  r.  No. I Br. 


137.35 


-15- 


' Now  61.3  - 35  = 26.3  vvhich  represents  the  number  Cos  of 

.0948  N.  bromine  absorbed  by  .S3  grams  of  cod  oil. 

One  cc  of  normal  bromine  contains  .07992  grams  of  Brs 

therefore  the  following  gives  the  bromine  value 

36.5  X .0948  X .07992  s 86.53  the  bromine  absorption 
.23 

value  of  cod  oil  standing  in  contact  with  bromine  for  8 hours 
at  the  tem.perature  of  ice  and  salt . 

Since  the  previously  determined  iodine  value  is  the 
measure  of  the  fo  maximum  possible  absorption,  the  iodine  value 
is  calculated  in  terms  of  the  bromine  value. 

Results : 

The  following  present  the  results  by  varying  the  time 
and  the  appended  curves  represent  the  same  in,  graphical  form. 


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- 35- 

The  preoeding  ta’-les  and  curves  for  the  various  fish  oils 
indicate  that  the  bromine  a";' sorption  values  of  these  oils  increase 
with  the  length  of  time  in  which  they  are  allowed  to  remain  in 
contact  with  the  bromine. 

In  general  the  rise  seems  to  take  place  rather  uniforii.ly, 
a few  ■‘^rea  fis  being  noted — but  since  the  ’whole  increase  is  over 
such  a small  range — this  might  be  due  to  slight  differences  in 
temperature,  or  a little  loss  in  bromine  because  of  its  volatility. 

The  oils  with  the  lower  iodine  values  seem  to  give  the 
most  consistent  results,  menhaden,  as  has  been  said  before,  giving 
the  most  trouble  along  this  line,  pro’^ably  due  to  its  greater 
degree  of  unsaturation.  The  increase  in  this  case  is  also  more 
marked  with  time. 

CONCLUSIONS . 

1 . The  Bromine  absorption  values  of  fish  oils  as 
determined  vary  with  the  time  of  contact  with  bromine. 

2.  The  increase  with  time  is  related  to  the  unsaturation 
of  the  fat,  the  less  saturated  sho'ving  a more  marked  rise  than  the 
more  saturated  ones  . 

3.  The  conditions  which  come  nearest  the  time  of  true 
absorption  values  as  calculated  from  the  iodine  numbers  are  as 
represented  with  each  oil — 16  hours  at  -10^  C . 


-r^ 

^■’’•v  V^  . *•*’ 

:;.V'A^Vj.*,  V...  • ' ,-  •• 


. • * . ■ r7  ‘ ■ r-Jf  r ■ ' ‘ V-' 

■■•  ■ - 


■'-  -V 


